The aim of this proposal is to define the molecular mechanism of somatic hypermutation of immunoglobulin (Ig) genes. We have recently obtained conclusive evidence that V-region specific mutation of functional Ig genes can occur without ongoing rearrangement and at random chromosomal sites (O'Brien et al., submitted). Therefore, a special mutator must exist which alters previously rearranged Ig genes. The proposed work has the goal to determined the mechanisms responsible for the mutations. Specifically the following experiments are planned: 1) Identification of stably transformed cell lines with ongoing somatic mutations of Ig genes: A source of cells with ongoing mutations is required for the analysis of the enzymology involved in the mutations and to speed up the search for essential accessory sequences outside of the mutation targets. 2) Determination of whether the mutations are really VJ region restricted in situations where C-region mutations cannot be selected against: We will determine the exact regional limits of the mutations by sequencing nonfunctional K transgenes and the nonproductive H and L alleles of several myelomas and hybridomas where the productive alleles are known to have mutations in the V region. If these experiments show that the mutations are restricted to the V region and vicinity we will construct a K gene where a C region is inserted into the middle of the V region (VCV), to determine whether it is location or sequence that determines that normally only V regions are mutated. 3) Accessory DNA sequences outside of the mutator target which are required for the mutations: Is a V gene alone sufficient or are Ig promoters and/or Ig enhancers required? 4) Mutations due to recombination by short stretches of sequence from multiple donor genes will be investigated in transgenic mice with two different, but related and experimentally, distinguishable K genes. Exchange of sequences will be studied and the relative frequencies of recombinational and other mutations will be compared. 5) Nuclear extracts of mutating cells will be analyzed for the presence of proteins which specifically bind to V-region DNA. In future experiments it may then be possible to purify such proteins and to reconstruct a cell- free mutation sytem.